Paediatric cardiomyopathies
Herkert, Johanna Cornelia
DOI:
10.33612/diss.97534698
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Herkert, J. C. (2019). Paediatric cardiomyopathies: an evolving landscape of genetic aetiology and
diagnostic applications. Rijksuniversiteit Groningen. https://doi.org/10.33612/diss.97534698
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Chapter 9
Johanna C. Herkert*, Rowida Almomani*, Anna Posafalvi,Jan G. Post, Ludolf G. Boven, Paul A. van der Zwaag, Peter H.G.M. Willems, Ingrid H. van Veen-Hof, Judith M.A. Verhagen, Marja W. Wessels, Peter G.J. Nikkels, Liesbeth T. Wintjes, Maarten P. van den Berg, Richard J. Sinke, Richard J. Rodenburg, Klary E. Niezen-Koning, J. Peter van Tintelen, Jan D.H. Jongbloed
*These authors contributed equally
Journal of Medical Genetics 2019;doi: 10.1136/jmedgenet-2019-106330
Homozygous damaging SOD2 variant causes lethal
neonatal dilated cardiomyopathy
Abstract
Background Idiopathic dilated cardiomyopathy (DCM) is recognized to be a heritable disorder,
yet clinical genetic testing does not produce a diagnosis in >50% of paediatric patients. Identifying a genetic cause is crucial because this knowledge can affect management options, cardiac surveillance in relatives and reproductive decision-making. In this study, we sought to identify the underlying genetic defect in a patient born to consanguineous parents with rapidly progressive DCM that led to death in early infancy.
Methods & Results Exome sequencing revealed a potentially pathogenic, homozygous missense
variant, c.542G>T, p.(Gly181Val), in SOD2. This gene encodes superoxide dismutase 2 (SOD2) or manganese-superoxide dismutase, a mitochondrial matrix protein that scavenges oxygen radicals produced by oxidation-reduction and electron transport reactions occurring in mitochondria via conversion of superoxide anion (O2–•) into H
2O2. Measurement of hydroethidine oxidation showed a significant increase in O2−• levels in the patient’s skin fibroblasts, as compared with controls, and this was paralleled by reduced catalytic activity of SOD2 in patient fibroblasts and muscle. Lentiviral complementation experiments demonstrated that mitochondrial SOD2 activity could be completely restored on transduction with wild type SOD2.
Conclusion Our results provide evidence that defective SOD2 may lead to toxic increases in the
levels of damaging oxygen radicals in the neonatal heart, which can result in rapidly developing heart failure and death. We propose SOD2 as a novel nuclear-encoded mitochondrial protein involved in severe human neonatal cardiomyopathy, thus expanding the wide range of genetic factors involved in paediatric cardiomyopathies.
9
Introduction
Dilated cardiomyopathy (DCM) is characterized by left ventricular enlargement and systolic dysfunction that can lead to heart failure and sudden cardiac death. DCM is a major cause of childhood mortality and the most common indication for heart transplantation in children.1 Annual incidence of DCM in children is approximately 0.57/100,000 and is even higher in children below 12 months of age (8.34/100,000).1 Recognition of idiopathic DCM as a familial disease2 has led to the discovery of more than 60, mostly adult-onset, DCM-associated genes that encode transcription factors and cytoskeletal, sarcomeric, ion transport, nuclear membrane and mitochondrial proteins.3-8 Current next-generation gene panel testing yields a genetic diagnosis in ̴ 37-50% of paediatric cases.9-11
Nevertheless, knowledge of the underlying genetic causes of congenital or neonatal DCM remains limited. In these cases, DCM often occurs in the context of a malformation syndrome or disorders of metabolism and/or energy production, which are generally autosomal recessively inherited. Gene panels that target ‘adult-onset’ DCM genes are therefore less appropriate for severe infantile forms of DCM.12 In contrast, exome sequencing (ES) (or genome sequencing) provides a powerful platform for novel disease gene discovery, particularly in congenital or neonatal cardiomyopathy. Successful application of ES to identify novel pathogenic variants in paediatric DCM has been recently demonstrated.13-17
Here, we report a neonate with lethal DCM born to consanguineous parents. ES identified a homozygous, damaging variant in SOD2, which encodes superoxide dismutase 2 (SOD2) or manganese-superoxide dismutase (MnSOD). Consistent with the function of SOD2, the resulting missense variant was shown to affect the catalytic activity of the protein, leading to excess oxygen radical levels that can have strongly damaging effects in the neonatal heart. Lentiviral gene rescue restored superoxide dismutase activity. Our findings suggest a role for
SOD2 in inherited cardiomyopathy and add a novel gene to the still-expanding list of genes
Patients and methods
Patient
Medical records of patient X:1 were carefully reviewed. Cardiac evaluation of her parents and siblings included echocardiography and electrocardiography (ECG). Diagnostic criteria for DCM were left ventricular end-diastolic and/or systolic short-axis chamber dimension Z-score ≥2.0 and fractional shortening or left ventricular ejection fraction >2 SD below the mean for age in the absence of abnormal loading conditions sufficient to cause global systolic impairment.18 Genomic DNA of patient X:1 and her parents was extracted from peripheral blood. Fibroblast cells were established from a postmortem skin biopsy of patient X:1. Enzymatic activity of the five respiratory chain complexes and citrate synthase were determined in an enriched mitochondrial fraction of fibroblasts by spectrophotometric assays, as described previously.19,20 The parents provided informed consent for the DNA studies and diagnostic procedures. The UMCG ethical committee approved the study.
Histology
Heart samples obtained at autopsy from left and right ventricular wall and septum were formalin-fixed and paraffin-embedded using standard clinical laboratory protocols. Heart (left ventricle) and skeletal muscle samples were frozen and stored at -80°C.
ES, variant filtering and interpretation
ES was performed using a previously described parent–offspring trio approach.9,13 Briefly, the exome was captured using the Agilent SureSelect XT Human All Exon V6 kit (Agilent, Santa Clara, CA, USA). Exome libraries were sequenced on an Illumina NextSeq500 instrument (Illumina, San Diego, CA, USA) with 151 bp paired-end reads. Sequence reads were aligned to the GRCh37/ hg19 reference genome using BWA version 0.7.5a. Local realignment of insertions/deletions and base quality score recalibration were performed using the Genome Analysis Toolkit Haplotype Caller, version 3.7. Variants were annotated using a custom diagnostic annotation pipeline, then filtered for rarity excluding those with a minor allele frequency (MAF) >1% for heterozygous variants in an autosomal dominant inheritance model and those with MAF >2% for homozygous or compound heterozygous variants in an autosomal recessive inheritance model. After exclusion of variants in known cardiomyopathy-related and nuclear-encoded mitochondrial genes, we selected variants found in homozygous state in the patient and in heterozygous state in both parents.
Variant pathogenicity was assessed using data from the Agilent Alissa clinical informatics platform (Cartagenia, Leuven, Belgium) and Alamut Visual software (Interactive Biosoftware, Rouen, France), a gene browser that integrates missense prediction tools, allele frequencies from different population databases (1000 Genomes Project21, Genome of the Netherlands22,
9
GnomAD23 and disease-specific databases (HGMD, ClinVar, LOVD)) and mRNA splicingprediction tools. Literature and allele frequencies from our in-house population database were also used for pathogenicity determinations. Classification of variants was based on American College of Medical Genetics and Genomics guidelines.24
Sanger sequencing
Sanger sequencing was performed to confirm the presence/absence of the SOD2 variant in patient X:1, her parents and her siblings. We also screened all exons and exon/intron junctions of SOD2 in79 unrelated patients with childhood-onset cardiomyopathy and 161 adult patients with DCM (primers available upon request). PCR was performed by using AmpliTaq Gold PCR Master Mix (Invitrogen Life Science Technologies, Carlsbad, CA, USA) following the official protocol. Resulting fragments were sequenced by the Applied Biosystems 96-capillary 3730XL system (Carlsbad, CA, USA).
Splicing analysis
RNA was isolated from cultured fibroblasts from patient X:1 and fibroblast cells from a healthy control. Cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% foetal bovine serum, 1% penicillin/streptomycin, 1% glucose and 1% glutamax in a humidified atmosphere of 5% CO2 and atmospheric O2 at 37°C. RNA was extracted using the RNeasy Mini Kit (QIAGEN, Venlo, the Netherlands). cDNA was synthesized from 500 ng of total RNA by RevertAid RNaseH-M-MuLV reverse transcriptase in a total volume of 20μl according to the supplier’s protocol (MBI-Fermentas, St. Leon-Rot, Germany).
To investigate whether the SOD2 c.542G>T variant affects mRNA splicing, we performed reverse-transcription PCR with gene-specific primers designed to amplify the exon expected to be affected by the variant and flanking sequences (primers available upon request). The resulting PCR products were examined by 2% agarose gel electrophoresis and subsequently analysed by Sanger sequencing. To test whether mRNA is degraded by nonsense-mediated mRNA decay (NMD), fibroblasts were treated with the NMD inhibitor cycloheximide for 4.5 hours, followed by RNA analysis using the same procedures used for RNA from untreated cells.
Measurement of hydroethidine-oxidizing reactive oxygen species levels
Fibroblasts, cultured to 70% confluence, were incubated in HEPES-Tris medium containing 10 μM hydroethidine (HEt; Molecular Probes, Leiden, the Netherlands) for 10 min at 37°C. Within the cell, two HEt oxidation products are formed under oxidative conditions: 2-hydroxyethidium (2-OH-Et), the sole reaction product of HEt and O2−•, and ethidium (Et).25 Both products are positively charged and fluoresce when excited at 490 nm. The reaction was stopped by thoroughly washing the cells with phosphate-buffered saline to remove excess HEt. For quantitative analysis of HEt oxidation products as a measure of HEt oxidizing reactive oxygen
species (ROS) levels, coverslips were mounted in an incubation chamber placed on the stage of an inverted microscope (Axiovert 200 M; Carl Zeiss, Jena, Germany) equipped with a Zeiss ×40/1.3 NA Fluar oil-immersion objective. HEt oxidation products were excited at 490 nm using a monochromator (Polychrome IV; TILL Photonics, Gräfelfing, Germany). Fluorescence emission was directed using a 525DRLP dichroic mirror (Omega Optical, Brattleboro, VT, USA) through a 565ALP emission filter (Omega Optical) onto a CoolSNAP HQ monochrome charge-coupled device camera (Roper Scientific, Vianen, the Netherlands). The image-capturing time was 100 ms and 10 fields of view per coverslip were routinely analysed.
Superoxide dismutase assay
Superoxide dismutase was measured using the Sigma SOD assay kit (19160-KT-F) following the manufacturer’s procedures. This assay is based on the reduction of the tetrazolium salt WST-1 by superoxide into a formazan dye that can be monitored spectrophotometrically at 440 nm. Measurements were performed in mitochondria-enriched fractions prepared from cultured skin fibroblasts. SOD activity assays were performed in two independent experiments, each performed in duplicate (four measurements per sample in total). To confirm specific SOD2 residual activity in muscle, superoxide dismutase activity was measured in patient-derived muscle cells using the ENZO SOD assay kit (ADI-900-157) according to the manufacturer’s procedures. Cu/ZnSOD (SOD1) activity was inhibited by 20 mM potassium cyanide (KCN). MnSOD (SOD2) activity is not inhibited at these levels of KCN. Measurements were performed in triplicate in a single-cell suspension prepared from patient and control muscle tissue.
Proteomics analysis
To quantify the amount of SOD2 protein in skin fibroblasts, we used QconCAT technology in combination with mass spectrometry according to Wolters et al.26
Lentiviral complementation
A plasmid containing full length hSOD2 cDNA (NM_001024466.1) was purchased from PlasmID Repository, Harvard, USA (clone HsCD00042604). The open reading frame was recombined into the pLenti6.2V5-DEST destination vector (Invitrogen) using the Gateway LR clonase II enzyme mix (Invitrogen). The production of lentiviral particles containing the SOD2 cDNA and transduction of patient-derived fibroblasts was performed as described previously.27 As a negative control, cells were transduced by the gene encoding green fluorescent protein.27
SOD2 3D structure
We used HOPE software28 to predict the effect of the SOD2 missense variant on 3D protein structure and the Uniprot protein database (www.uniprot.org) to search for known functional features within the mitochondrial superoxide dismutase [Mn] protein (accession number: P04179) in the region affected by the genetic variation.
9
Results
Clinical findings
The female proband (patient X:1) was delivered by secondary caesarean section for breech presentation, prolonged labour and meconium-stained amniotic fluid at 39 weeks of gestation. Prenatal ultrasound at gestational age 19+4 weeks was normal. Apgar scores were 2, 3 and 9 at 1, 5 and 10 minutes, respectively. Birth weight was 2240 g (<p2.3), length at birth was 49 cm (p25) and head circumference was 33.0 cm (p5). Umbilical artery pH was 7.14 (n=7.37-7.45) with a base excess of -4 mmol/L indicative of metabolic acidosis. Cerebral function monitoring was normal, but cerebral echography showed intraventricular septa. The day after birth, she presented with apnoeas, poor circulation and mild tachycardia. Mean arterial pH was 7.38 (n=4), haemoglobin 8.6 mmol/L (n=8.3-12.4 mmol/L) and blood lactate 10.4 mmol/L (n=0.0-2.2 mmol/L). A chest X-ray was normal. Echocardiography showed a structurally normal heart, but left ventricular function seemed poor. Cardiac troponin and B-natriuretic peptide were elevated at 0.28 μg/L (n<0.16 μg/L) and 2819 pmol/L (n<35 pmol/L), respectively.
On the third day after birth, patient X:1 developed acute cardiogenic shock with oliguria, metabolic acidosis with increased blood lactate of 18 mmol/L and poor body circulation. ECG showed signs of myocardial ischemia, and QTc was slightly prolonged (QTc 442-474 ms). Echocardiography revealed DCM with severe biventricular dilation and decreased left ventricular ejection fraction. Frequent ventricular extrasystoles and tachycardia were observed. At day 4 she developed ventricular fibrillation and was successfully defibrillated four times. However, there was no improvement of ventricular function despite intensive treatment with dopamine, milrinone and adrenalin, and she died at age 4 days. Biochemical studies at day 3 showed increased amino acids in blood, including proline and alanine, and increased organic acids in urine, including 3-methylglutaconic acid. No underlying cause for her DCM was identified despite extensive pre- and post-mortem diagnostic workup that included viral serology in blood, sputum and faeces, metabolic testing (including for Pompe disease) and copy number variant analysis (Agilent 180K oligo-array). As recommended in children with 3-methylglutaconic aciduria type IV, sequencing of mitochondrial DNA and the nuclear genes POLG, RYR1 and
SUCLA2 was performed as previously described.29 Additionally, the MYL2, MYH7, LMNA and
DES genes were assessed for (biallelic or de novo) variants because these genes were at that time
known to putatively carry variants associated with early-onset DCM. However, no pathogenic variants were found.
The parents were of Dutch origin. Genealogical evaluation revealed a distant relationship between the parents 6 to 8 generations ago (Figure 1A). Three-generation family history was negative for heart failure. The paternal grandfather underwent coronary bypass surgery. Cardiac evaluations in the parents (aged 27 and 29 years) and siblings, both evaluated at age
1 week, were normal.
A B
C
Figure 1. Pedigree, heart and brain imaging of proband. (A) Pedigree of Dutch family with a child
with severe, lethal DCM in whom autosomal recessive inheritance was expected due to consanguinity. Squares=males, circles=females, solid symbol=affected, mut=mutant allele, wt= wild-type allele. (B) Macroscopic section of the heart, showing severe left ventricular (LV) and right ventricular (RV) chamber dilation. (C) Macroscopic cross-section of the brain showing subependymal cysts (SECs) (arrowheads).
Histopathological examination and enzymatic activity
Examination of the heart at autopsy revealed severe dilatation of both ventricles compared with an age-matched control (Figure 1B), without fibroelastosis, interstitial fibrosis, cardiomyocyte hypertrophy or disarray. Electron microscopy was not performed. Cardiac weight (20.6g) was consistent with 41 weeks gestation (and relatively heavy as expected with DCM developing in the neonatal period30), while the weights of other organs were low and consistent with 32-33 weeks gestation. There were no signs of glycogen storage or intracellular lipid vacuoles in hepatocytes, cardiomyocytes, skeletal muscle or tubular cells of the kidney. Skeletal muscle showed no abnormalities, and there were no indications of disorders of fatty acid oxidation or mitochondrial disease although these could not be fully excluded by histological examination.
9
Intracranial examination showed small cerebral subependymal cysts, some with smallsepta surrounded by macrophages, suggestive of hypoxic/ischaemic damage that occurred in utero (Figure 1C). Neurons in the brainstem, cerebellum, basal ganglia, thalamus, hippocampus and cerebral cortex showed signs of recent hypoxia. Actin, dystrophin, sarcoglycan, dystroglycan, dysferlin, caveolin-3, merosin, myosin and spectrin-1 staining were normal in skeletal muscle, and stains for the respiratory chain complexes, including succinate dehydrogenase and cytochrome c oxidase, in frozen material from heart and skeletal muscle were normal (data not shown).
Exome sequencing
To look for potential disease-causing variants, ES targeted all exons and exon/intron junction sequences of known genes in the human genome. Using the filtering pipeline discussed earlier, we excluded likely pathogenic or pathogenic variants in 312 genes related to (childhood-onset) cardiomyopathy and 355 nuclear-encoded mitochondrial genes (Supplemental Table 1). Assuming homozygosity by descent of an autosomal recessive variant to be the likely cause of DCM in patient X:1, we selected for homozygous variants in concordance with autosomal recessive inheritance. This identified eight homozygous variants with allele frequencies <2% in the public databases (Supplemental Table 2). Only one, c.542G>T in SOD2, was classified as likely pathogenic, and it was predicted to cause a substitution of a highly evolutionarily conserved glycine at position 181 with valine (p.(Gly181Val), SOD2, NM_000636.3) at the protein level (Figure 2). The p.(Gly181Val) missense variant was present in 1 of 248,906 control alleles in GnomADv2.2.1 (absent homozygously), absent in control individuals from GoNL and predicted to be damaging (e.g., phyloP 6.18, Grantham-score 109). Notably, this variant is located in the longest autosomal homozygous region on chromosome 6, between rs378512 and rs9458499 (2.76 Mb; UCSC Genome Browser, build hg19) (data not shown). We validated this result by Sanger sequencing and verified segregation of the variant in all available family members. Both parents were heterozygous for the variant. DNA testing in saliva of the unaffected brother (X:2) excluded the SOD2 variant in homozygous state and chorionic villous biopsy performed in the third pregnancy showed absence of the variant in the foetus (X:3).
Reverse transcription polymerase chain reaction (RT-PCR) and SOD2 protein quantification
Two of five mRNA splicing prediction tools in the Alamut software indicated a possible new donor site. To further assess the pathogenicity of this variant at RNA level, we performed RT -PCR analysis. RT-PCR product analysis of RNA isolated from patient-derived fibroblasts cultured both with and without cycloheximide showed a transcript of only wild-type size, indicating that this variant did not affect splicing. Quantitative protein analysis with targeted quantitative Liquid Chromatography-Mass Spectrometry-Selected Reaction Monitoring (LC-MS-SRM) using stable isotope-labelled concatemers26 confirmed the presence of the SOD2 protein (Figure 3A).
We therefore concluded that the SOD2 missense variant probably results in a conformational change that affects protein folding or flexibility, protein-protein or protein-DNA interaction or the activity of the Manganese (Mn)-cofactor binding domain, rather than resulting in absent or decreased expression of SOD2.
*
H. sapiens mutated P L L V I D V W E H. sapiens wildtype P L L G I D V W E P. troglodytes P L L G I D V W E R. norvegicus P L L G I D V W E M. musculus P L L G I D V W E C. familiaris P L L G I D V W E O. anatinus P L L G I D V W E G. gallus P L L G I D V W E X. tropicalis P L L G I D V W E T. nigroviridis P L L G I D V W E D. rerio P L L G I D V W E D. melanogaster P L F G I D V W E C. elegans P L F G I D V W E Bakers yeast P L V A I D A W EA
B
Figure 2. Confirmation and properties of SOD2 variant c.542G>T, p.(Gly181Val). (A) Sanger sequencing
verified the presence of the SOD2 variant c.542G>T, p.(Gly181Val) in the affected patient (bottom) compared with control (top) and in heterozygous form in her parents (not shown). (B) Conservation of Gly181 and surrounding residues.
9
SOD2 mutational screening
Sanger sequencing of SOD2 in a cohort of 79 unrelated patients with childhood-onset cardiomyopathy and 161 adult patients with DCM and NGS-gene-panel–based sequencing of this gene in 1,150 mostly adult cardiomyopathy patients revealed no pathogenic or likely pathogenic SOD2 variants.
Effect of the SOD2 variant on intracellular superoxide (O2−•) levels
HEt was used as a probe to measure the intracellular levels of HEt-oxidizing ROS levels, including O2−•, in patient fibroblasts. The oxidation levels of HEt measured in our in vitro assay indicated a significant increase of O2−• levels in patient fibroblasts comparable in order of magnitude to that seen in complex I–deficient fibroblasts (Figure 3B). Because mitochondrial respiratory chain enzyme activities (complexes I–V) were normal in mitochondria-enriched fractions from skin fibroblasts, the significant increase in O2−• levels we observe probably resulted from impaired SOD2 enzyme activity.
Effect of identified variant on SOD2 activity
MnSOD (SOD2) enzyme activity in patient-derived muscle cells was investigated in the cell pellet fraction. Total SOD activity was shown to be reduced in this cell pellet fraction when compared with a control (49 U/mg protein versus 58 U/mg protein, respectively). These data suggest that enzymatic SOD2 activity was decreased in the patient, despite higher levels of SOD2 protein. To confirm this, the same experiment was performed after inhibition of SOD1 and SOD3 by potassium cyanide (KCN) (20 mM). Our data indicated a residual SOD2 activity of 40% in the patient; however, our data also suggested different inhibition levels between the patient and control; therefore, we could not draw firm conclusions from this experiment.
Rescue of superoxide dismutase activity by lentiviral complementation
To further investigate whether the genetic defect in SOD2 was responsible for the increased superoxide levels, we stably transfected patient-derived fibroblasts with wild-type SOD2 cDNA and monitored the superoxide activity in whole-cell lysates and a mitochondria-enriched fraction. Our results confirmed that the observed superoxide dismutase activity was clearly reduced (0.46 ± 0.04 U/mg in mitochondria-enriched fraction from the patient versus 0.80-1.80 U/mg in controls; Table 1). In comparison, the total superoxide dismutase activity in whole-cell lysates was very similar (results not shown). On transduction of the patient cells with wild-type
SOD2 cDNA, mitochondrial superoxide dismutase activity was completely restored (Table 1),
A
B
Figure 3. (A) Quantitative protein analysis in mitochondria-enriched fractions of patient-derived skin
fibroblasts by targeted quantitative LC−MS−SRM using stable isotope-labelled concatemer. Blue peak represents the isotope-labelled standard. Red peak represents the same endogenous peptide. A known amount of the isotope-labelled standard was added to the protein sample, allowing the amount of the endogenous peptide (and therefore protein) to be calculated. The SOD2 protein is at least two-times higher compared with controls (patient 27.6 fmol/µg compared with two different controls 11.7 ± 1.3 fmol/μg total mito protein). (B) The oxidation of hydroethidine analysis shows a significant increase of ROS OR (O2•−) level in the fibroblasts of the patient compared with control fibroblasts. LC-MS-SRM=Liquid
Chromatography-Mass Spectrometry-Selected Reaction Monitoring, Mito=mitochondria, Nuc=nucleus, SOD2=superoxide dismutase 2.
9
Table 1. Rescue of superoxide dismutase 2 activity in patient fibroblasts by lentiviral transductionof wild-type SOD2. SOD2 activity (U/mg protein) Patient X:1 0.46 ± 0.04 Patient X:1 + GFP 0.37 ± 0.15 Patient X:1 + SOD2 1.74 ± 0.06 Control range (n=6) 0.80-1.80*
Values in bold are below the control range. GFP=green fluorescent protein. *average 1.23
SOD2 3D structure: the effect of the p.(Gly181Val) variant
Using the HOPE software, we retrieved 3D-structure information for the SOD2 protein through the WHAT IF Web services, the Uniprot database and a series of DAS-servers to predict the effect of the p.(Gly181Val) variant on the protein structure. The Gly181 residue is part of a Mn/ iron superoxide dismutase domain important for superoxide dismutase activity (oxidoreductase activity) and metal ion binding. According to the Uniprot database, four important amino acid residues (His50, His98, Asp183 and His187) are involved in the formation of the Mn-binding pocket that binds the Mn cofactor of the enzyme (accession number: P04179). Interestingly, the aspartic acid residue of key importance (Asp183) is only two amino acids away from the Gly181 residue that was mutated in our patient. The increased size of the mutant residue is predicted to disturb the core structure of the Mn/iron superoxide dismutase domain and, in consequence, the catalytic activity of the enzyme (Figure 4A, B).
Discussion
We have demonstrated, for the first time, an association between a damaging variant in SOD2 and DCM in humans. ES in a patient with neonatal DCM born to consanguineous parents revealed a novel homozygous missense variant, c.542G>T; p.(Gly181Val), in SOD2. This gene encodes a mitochondrial superoxide scavenger enzyme that protects cells from ROS damage by detoxifying oxygen radicals such as O2–• to yield O
2 and hydrogen peroxide,which is then metabolized into H2O by glutathione peroxidase31(Figure 4C). The SOD2 variant in our patient was not identified as homozygous in any individual in the GnomAD database and was predicted to affect protein function. It is located in the functionally important C-terminal Mn/ iron superoxide dismutase region of the protein, very close to one of the four histidine/aspartic acid residues involved in binding the Mn co-factor. Significant differences between the size and physico chemical characteristics of the wild-type glycine, which is the smallest amino acid residue and provides flexibility for enzyme active sites, and the mutant valine residue are predicted to disturb the core structure in this crucially important domain. This suggests that the identified variant would significantly affect the protein’s stability and/or activity.
We performed several analyses to evaluate the predicted effect on SOD2 stability/activity. RNA studies showed a stable SOD2 transcript not subject to nonsense-mediated decay. Protein quantification by LC-MS-SRM showed increased SOD2 levels, which can be explained by the upregulation of SOD2 expression in response to oxidative stress.33 HEt oxidation measurements in patient fibroblasts indicated a significant increase in levels of O2−•, one of the major ROS substrates of SOD, comparable in order of magnitude to the levels seen in complex-I-deficient fibroblasts.25,34 Since no deficiency was seen in any of the mitochondrial respiratory chain complexes I-V, the significant increase in O2−• is likely explained by the pathogenic effect of the c.542G>T; p.(Gly181Val) SOD2 variant on the function of the encoded enzyme, leading to increased oxidative stress and accumulation of damaging oxygen radicals in the cells. Despite increased SOD2 expression, decreased SOD2 activity was measured in patient-derived skin fibroblasts and muscle. Together with our successful rescue experiments, where patient fibroblasts were complemented with wild-type SOD2 cDNA, these data support a role for mutated SOD2 in the pathogenesis of DCM in this patient.
9
A B C Respiratory chain complexesMitochondrion
Outer membraneO
2.-H
2O
2 SOD22H
2O
GPxO
2 Inner membrane Matrix DNA NO. ONOO- NADH e- Fe2+H
2O
2 .OH
H
2O + O
2 Catalase2H
2O
GPx MPOClO
-O
2.-H
2O
2 SOD1◀ Figure 4. (A) Overview of the SOD2 protein in ribbon-presentation. (B) Zoom-in on the part of the manganese/iron superoxide dismutase domain where the mutated residue is located. The protein backbone is shown in grey, the side chains of both the wild-type in green and the mutant residue in red. The mutant residue is larger than the wild-type residue, which might disturb the core structure of this domain and affect the catalytic activity of the enzyme. (C) Mitochondrial free radical generation and its catabolism. Highly reactive superoxide anions are mainly produced in mitochondria and by the cytosolic xanthine oxidase, plasma membrane-associated NADPH-oxidase complex (NOX) and the cytochrome p450 monooxygenases, which are present mainly in the endoplasmatic reticulum. Superoxide anions can either react with nitric oxide to generate the strong oxidant peroxynitrite (ONOO-) or be degraded by
superoxide dismutase (SOD) into the less reactive species hydrogen peroxide (H2O2). Hydrogen peroxide can then be catabolized by glutathione peroxidase (GPx) or catalase, react with Fe2+ to form hydroxyl
radicals via the Fenton reaction or be degraded by the myeloperoxidase (MPO) into hypochlorite (ClO-),
another source of hydroxyl radicals (adapted from Ref.32).
SOD2 belongs to the Mn/iron superoxide dismutase family, one of the primary families of antioxidant enzymes in mammalian cells. These antioxidant enzymes protect cells from the damage caused by ROS. In eukaryotic cells, there are three SOD homologs: cytosolic Cu/ ZnSOD (SOD1), Mn/FeSOD (manganese superoxide dismutase 2; SOD2) and extracellular SOD3. SOD2 is a mitochondrial matrix protein that forms a homotetramer and binds one Mn ion per subunit.
ROS are formed during normal mitochondrial metabolism as byproducts of oxidative phosphorylation, and cells self-regulate their ROS levels by producing antioxidant enzymes.35 Deficiency of one of the antioxidant enzymes, such as SOD2, may lead to accumulation of ROS, particularly during times of environmental stress. Excessive amounts of ROS can damage cellular proteins, DNA and lipids.36 As is commonly observed in mitochondrial disorders, ROS-mediated oxidative stress can affect any organ at any age, but it particularly affects organs with high energy demand such as the heart and brain.37
Oxidative stress and SOD2 gene polymorphisms have been associated with premature aging38, cancer39, neurological disorders40,41, diabetes42,43 and cardiovascular diseases including hypertension and atherosclerosis.44-46 Oxidative stress and disturbed mitochondrial respiratory function are also known to play a substantial role in the development of heart failure.47 Hiroi and colleagues found an increased frequency of the SOD2-16VV genotype in Japanese patients with non-familial idiopathic cardiomyopathy and suggested that this polymorphism contributes to the susceptibility to non-familial idiopathic cardiomyopathy.48 In addition, anthracyclin-induced cardiomyopathy is believed to be a side effect of superoxide radical accumulation that leads to the induction of mitochondrial dysfunction in the heart.49 This phenotype was successfully rescued in transgenic mice by overexpression of SOD250, further underscoring the cardio-protective role of this enzyme.
9
The role of defective SOD2 protein in the pathophysiology of DCM has previously beendemonstrated in mice. Homozygous Sod2 knockout (Sod2-/-) mice (outbred background, CD1) lacking exon 3, which results in the complete loss of SOD2 enzyme activity, display cardiomyopathy at age 5 days. Similar to our case, these SOD2-deficient mice had enlarged hearts with a dilated left ventricular cavity.51 Given our patient’s early death at age 3 days, it is perhaps not surprising that we did not observe the endocardial fibrosis or cardiomyocyte hypertrophy seen in mutated mice, which usually die at age 10 days.51 Another strain of Sod2-/- mice (SOD2m1BCM/SOD2m1BCM, hybrid background) was shown to be able to survive for up to 18 days, with only 10% developing severe DCM.52 In these (older) mice, electron microscopy of the brain and spinal cord at postnatal day 10 revealed degenerative injury to large CNS neurons, particularly in the basal ganglia and brainstem. Both SOD2-deficient mice strains develop lipid depositions in the liver, as shown at autopsy. Neither degenerative abnormalities nor fatty accumulation in the liver were observed in our patient, although she may have died too early to develop these features. Examination of her brain did reveal extensive white matter damage, most likely due to hypoxic/ischaemic-induced changes in utero. We hypothesize that this resulted from severe oxidative stress due to SOD2 deficiency, but cannot exclude birth asphyxia and/or poor circulation as contributing factors. Both SOD2-deficient mouse strains were severely growth-restricted compared with their littermates and showed metabolic acidosis, resembling our patient. Interestingly, further characterisation of the metabolic defects of the
Sod2-/- mice showed large amounts of 3-methylglutaconic, 2-hydroxyglutaric, 3-hydroxy-3
methylglutaric and 3-hydroxyisovaleric acids in urine53, as also seen in our patient. Respiratory chain deficiencies were not detected in our patient, a finding which is compatible with the animal model where histological and ultrastructural evidence of mitochondrial injury has been detected predominantly in older mice (P8-P18).51,52
Using QconCAT proteomics, we observed increased SOD2 protein levels in patient fibroblasts compared with controls. This mutated protein showed approximately 40% residual enzyme activity in skin fibroblasts (Table 1). In contrast, no SOD2 activity was detected in the homozygous mutant mice.51 This residual enzyme activity may explain the phenotypic differences between the mouse models and our patient. Increased labour-induced stress response 54,55 may have contributed to extreme metabolic disruption in our patient, although the exact disease mechanism leading to her dramatic disease course and early death remains unknown.
Conclusion
This study is the first to demonstrate a homozygous damaging variant in SOD2 as a cause of human DCM. Our results highlight a potential role for reduced SOD2 activity and abnormally elevated levels of oxidative stress in the pathogenesis of DCM. Together with results from previously published SOD2 animal studies, our findings suggest a model for mitochondrial oxidative damage in which superoxide-induced injury compromises mitochondrial function and
accelerates damage to cells and tissues with high energy demand, including cardiac myocytes. The beneficial effects of antioxidants on heart function and muscle fatigue seen in SOD2-deficient mice56,57 may lead to new therapeutic strategies, but further studies are needed to unravel the role of this enigmatic kinase in cardiac function and pathologic remodelling.
Acknowledgements
We thank the parents and siblings of the patient for participating in this study; Herma Renkema, Marion Ybema-Antoine, Maaike Brink, Frans van den Brandt and Sander Grefte for technical assistance;Karin Wolters for making the graphs of the QconCAT proteomics; and Kate McIntyre for editing the manuscript.
Conflict of Interest
PHGMW is scientific advisor of Khondrion, Nijmegen, the Netherlands. This SME had no involvement in the data collection, analysis and interpretation, writing of the manuscript and in the decision to submit the manuscript for publication.
Funding
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Supplemental Information
Supplemental Table 1. Likely pathogenic or pathogenic variants in these genes were excluded in patient X:1.
Gene panel mitochondrial diseases Gene panel paediatric cardiomyopathy
AARS2 MSTO1 AARS2 LIAS
ABAT MTFMT ABCC6 LMNA
ACAD9 MTO1 ABCC9 MAP2K1 ACO2 MTPAP ACAD8 MAP2K2 AFG3L2 NARS2 ACAD9 MGME1
AGK NAXE ACADS MIB1
AIFM1 NDUFA1 ACADVL MLYCD ALDH1B1 NDUFA10 ACTA1 MRPL3 ANO10 NDUFA11 ACTC1 MRPL44 APOPT1 NDUFA12 ACTN2 MRPS22 APTX NDUFA13 ADCY5 MTCP1 ATAD3A NDUFA2 AGK MTO1 ATAD3B NDUFA3 AGL MURC ATP13A2 NDUFA4 AGPAT2 MUT ATP5F1A NDUFA5 AHCY MYBPC3 ATP5F1B NDUFA6 AIP MYH6 ATP5F1C NDUFA7 ALG1 MYH7 ATP5F1D NDUFA8 ALG6 MYL2 ATP5F1E NDUFA9 ALMS1 MYL3 ATP5IF1 NDUFAB1 ALPK3 MYLK2 ATP5MC1 NDUFAF1 ANKRD1 MYOT ATP5MC2 NDUFAF2 ANKRD11 MYOZ1 ATP5MC3 NDUFAF3 ANKS6 MYOZ2 ATP5ME NDUFAF4 ANO5 MYPN ATP5MF NDUFAF5 APOPT1 NAGA ATP5MG NDUFAF6 ARSB NAGLU ATP5MGL NDUFAF7 ASNA1 NDUFA1 ATP5PB NDUFB1 ATP5E NDUFA11 ATP5PD NDUFB10 ATP6V1B2 NDUFAF1 ATP5PF NDUFB11 ATPAF2 NDUFAF2 ATP5PO NDUFB2 BAG3 NDUFAF3 ATP5S NDUFB3 BBS2 NDUFAF4 ATPAF1 NDUFB4 BCS1L NDUFAF5 ATPAF2 NDUFB5 BOLA3 NDUFB11
9
Supplemental Table 1. Continued.Gene panel mitochondrial diseases Gene panel paediatric cardiomyopathy
BCS1L NDUFB6 BRAF NDUFB3 BOLA1 NDUFB7 BRCA2 NDUFB9 BOLA2 NDUFB8 BRCC3 NDUFS1 BOLA3 NDUFB9 BRIP1 NDUFS2 C12orf65 NDUFC1 BSCL2 NDUFS3 C19orf12 NDUFC2 C10ORF2 NDUFS4 C19orf70 NDUFS1 CALR3 NDUFS6 C1QBP NDUFS2 CASQ1 NDUFV1 CA5A NDUFS3 CAV1 NDUFV2 CARS2 NDUFS4 CAV3 NEBL CEP89 NDUFS5 CDKN1C NEU1 CHCHD10 NDUFS6 CHKB NEXN CHKB NDUFS7 CHRM2 NF1 CLPB NDUFS8 CISD2 NKX2-5 CLPP NDUFV1 COA5 NPPA COA1 NDUFV2 COA6 NRAS COA3 NDUFV3 COG7 NSD1 COA5 NFS1 COL7A1 NUBPL
COA6 NFU1 COQ2 PALB2
COA7 NSUN3 COQ4 PCCA
COASY NUBPL COX10 PCCB
COQ2 OGDH COX14 PDGFRA
COQ4 OPA1 COX20 PDLIM3 COQ5 OPA3 COX6B1 PET100 COQ6 OXA1L COX7B PEX1 COQ7 PANK2 CPT1A PEX10 COQ8A PARS2 CPT2 PEX11B
COQ8B PC CRYAB PEX12
COQ9 PDHA1 CSRP3 PEX13 COX10 PDHB CTNNA3 PEX14 COX14 PDHX D2HGDH PEX16 COX15 PDK1 DCAF8 PEX19 COX20 PDK2 DCHS1 PEX2
COX4I1 PDK3 DES PEX26
COX4I2 PDK4 DLD PEX3
COX5A PDP1 DMD PEX5
Supplemental Table 1. Continued.
Gene panel mitochondrial diseases Gene panel paediatric cardiomyopathy
COX6A1 PDSS2 DNAJC19 PEX7 COX6A2 PET100 DOLK PGM1 COX6B1 PET117 DPM3 PHYH COX6B2 PIGA DSC2 PIGT COX6C PITRM1 DSG2 PKP2 COX7A1 PMPCA DSP PLEC COX7A2 PMPCB DTNA PLEKHM2
COX7B PNPT1 DYSF PLN
COX7B2 POLG ELAC2 PMM2 COX7C POLG2 EMD PNPLA2 COX8A PPA2 ENPP1 POLG COX8C PRKAA1 EPG5 POMT1
CP PTRH2 ERBB3 POU1F1
CTBP1 PUS1 ERCC4 PPARG
CYC1 PYCR1 EYA4 PRDM16
CYCS PYCR2 FAH PRKAG2
DARS2 QRSL1 FANCA PRPS1 DCAF17 RARS2 FANCB PSEN1 DDHD1 RMND1 FANCC PSEN2 DES RNASEH1 FANCD2 PTPN11 DGUOK RRM2B FANCE QRSL1 DHTKD1 RTN4IP1 FANCF RAB3GAP2
DLAT SACS FANCG RAD51C
DLD SAMHD1 FANCI RAF1 DLST SARS2 FANCL RBCK1 DMAC1 SCO1 FANCM RBM20 DMAC2 SCO2 FASTKD2 RET
DNA2 SCP2 FBN1 RIT1
DNAJC19 SDHA FBXL4 RMND1 DNAJC3 SDHAF1 FHL1 RYR2 DNM1L SDHB FHL2 SCARB2
EARS2 SDHD FIG4 SCN5A
ECHS1 SERAC1 FKRP SCO1 ECSIT SFXN4 FKTN SCO2 EHHADH SLC19A2 FLNA SDHA ELAC2 SLC19A3 FLNC SDHAF1 ERAL1 SLC25A1 FOS SDHD
9
Supplemental Table 1. Continued.Gene panel mitochondrial diseases Gene panel paediatric cardiomyopathy
ETHE1 SLC25A10 FOXD4 SEPN1 FA2H SLC25A12 FOXRED1 SGCA FARS2 SLC25A13 FTO SGCB FASTKD2 SLC25A19 FXN SGCD FBXL4 SLC25A21 GAA SGSH FDX2 SLC25A22 GATA4 SHOC2 FDXR SLC25A24 GATAD1 SKI FH SLC25A3 GATB SLC19A2 FOXRED1 SLC25A32 GATC SLC22A5 FTL SLC25A4 GBE1 SLC25A20 FXN SLC25A42 GJA5 SLC25A3 GARS SLC25A46 GLA SLC25A4 GATB SLC39A8 GLB1 SLC2A10 GATC SLC52A2 GMPPB SLX4 GATM SLC52A3 GNAS SNAP29 GFER SPART GNPTAB SOD2
GFM1 SPATA5 GNS SOS1
GFM2 SPG7 GPC3 SPEG
GLRX5 SQSTM1 GPR101 SYNE1
GLUD1 STAT2 GSN SYNE2
GTPBP2 STXBP1 GTPBP3 TACO1 GTPBP3 SUCLA2 GYS1 TAZ HARS2 SUCLG1 HADH TBX20 HCCS SUCLG2 HADHA TCAP HIBCH SURF1 HADHB TERT
HLCS TACO1 HAMP TGFB1
HSD17B10 TANGO2 HBB TGFB3 HSPA9 TARS2 HCCS TMEM126A
HSPD1 TAZ HCN4 TMEM43
HTRA2 THG1L HFE TMEM70
IARS2 TIMM44 HFE2 TMPO IBA57 TIMM50 HGSNAT TNNC1 ISCA2 TIMM8A HPS1 TNNI3 ISCU TIMMDC1 HRAS TNNI3K KARS TK2 HSD17B10 TNNT2 LACTB TMEM126A HSPB6 TPI1 LARS2 TMEM126B IDH2 TPM1
Supplemental Table 1. Continued.
Gene panel mitochondrial diseases Gene panel paediatric cardiomyopathy
LIAS TMEM186 IDUA TPM3 LIPT1 TMEM65 IGF2 TRNT1 LIPT2 TMEM70 ILK TSFM
LONP1 TPK1 ISL1 TTN
LRPPRC TRIT1 ITGB1BP2 TTPA LYRM4 TRMT10C JPH2 TTR
LYRM7 TRMT5 JUP TXNRD2
MARS2 TRMU KCNH1 UBE2T MCUR1 TRNT1 KCNH2 UBR1
MDH2 TSFM KIF20A VCL
MECR TTC19 KLF1 VPS13A
MFF TUFM KRAS WFS1
MFN2 TWNK LAMA4 XK
MGME1 TXN2 LAMP2 XPNPEP3
MICU1 TYMP LDB3 YARS2
MICU2 UQCC1 MIEF2 UQCC2 MIPEP UQCC3 MPC1 UQCR10 MPV17 UQCR11 MRM2 UQCRB MRPL12 UQCRC1 MRPL3 UQCRC2 MRPL40 UQCRFS1 MRPL44 UQCRH MRPL57 UQCRQ MRPS16 VARS2 MRPS2 VPS13D MRPS22 WARS2 MRPS23 WDR45 MRPS34 YARS2 MRPS7 YME1L1 MRRF
Supplemental Table 2. Homozygous variants identified in patient X:1 (allele frequency <2%).
Gene
Chromo-some
Transcript cDNA HGVS protein Coding effect gnomAD No. of
homo-zygotes
CADD SIFT PolyPhen2 Splice prediction 2015 ACMG criteria Classifi-cation SLC4A10 2 NM_001354461.1 c.313+15C>T p.? 967/250898 5 3.416 NA NA No/ uncertain effect BP4 VUS
SOD2 6 NM_000636.3 c.542G>T p.(Gly181Val) nonsynonymous 1/248906=0 0 27.6 deleterious prob.
damaging No/ uncertain effect
PS3PM2PP3 LP
SLC22A1 6 NM_003057.2 c.1320G>A p.(Met440Ile) nonsynonymous 1972/282860 15 24 tolerated benign pos. new splice acceptor site BS1 LB PLG 6 NM_000301.3 c.1878-6T>C p.? 452/282844 0 1.539 NA NA No/ uncertain effect BP4 VUS
SMO 7 NM_005631.4 c.808G>A p.(Val270Ile) nonsynonymous 2003/282734 7 21 tolerated benign No/
uncertain effect
BS1BP4 LB
GCOM1 15 NM_001285900.3 c.933+13G>A p.? 1721/257566 26 1.488 NA NA No/ uncertain effect BS1BP4 LB RAB37 17 NM_175738.4 c.545+10_545+ 14dupGGGCA p.? 2638/282072 14 NA NA NA No/ uncertain effect BS1BP4 LB
UMODL1 21 NM_001199527.1 c.3248G>A p.(Ser1083Asn) nonsynonymous 2257/280756 12 24.5 tolerated prob. damaging
pos. new donor site
BS1 LB
PM2 was applied when a variant was absent or extremely rare (<0.004%) in large population cohorts, as proposed by ClinGen’s Inherited Cardiomyopathy Expert Panel (Kelly et al., 2018: PMID 29300372). Variants in autosomal recessive genes were classified as likely benign as they were reported >5 times in homozygous state in gnomAD. ACMG=American College of Medical Genetics,
CADD=Combined Annotation Dependent Depletion v1.4, gnomAD=Genome Aggregation Database v2.1.1, HGVS=Human Genome Variation Society, PolyPhen-2=Polymorphism Phenotyping v2, prob.=probably, pos.=possible, SIFT=Sorting tolerant from intolerant, NA=not available/unknown, LB=likely benign, VUS=variant of uncertain significance, LP=likely pathogenic.
9
Supplemental Table 2. Homozygous variants identified in patient X:1 (allele frequency <2%).Gene
Chromo-some
Transcript cDNA HGVS protein Coding effect gnomAD No. of
homo-zygotes
CADD SIFT PolyPhen2 Splice prediction 2015 ACMG criteria Classifi-cation SLC4A10 2 NM_001354461.1 c.313+15C>T p.? 967/250898 5 3.416 NA NA No/ uncertain effect BP4 VUS
SOD2 6 NM_000636.3 c.542G>T p.(Gly181Val) nonsynonymous 1/248906=0 0 27.6 deleterious prob.
damaging No/ uncertain effect
PS3PM2PP3 LP
SLC22A1 6 NM_003057.2 c.1320G>A p.(Met440Ile) nonsynonymous 1972/282860 15 24 tolerated benign pos. new splice acceptor site BS1 LB PLG 6 NM_000301.3 c.1878-6T>C p.? 452/282844 0 1.539 NA NA No/ uncertain effect BP4 VUS
SMO 7 NM_005631.4 c.808G>A p.(Val270Ile) nonsynonymous 2003/282734 7 21 tolerated benign No/
uncertain effect
BS1BP4 LB
GCOM1 15 NM_001285900.3 c.933+13G>A p.? 1721/257566 26 1.488 NA NA No/ uncertain effect BS1BP4 LB RAB37 17 NM_175738.4 c.545+10_545+ 14dupGGGCA p.? 2638/282072 14 NA NA NA No/ uncertain effect BS1BP4 LB
UMODL1 21 NM_001199527.1 c.3248G>A p.(Ser1083Asn) nonsynonymous 2257/280756 12 24.5 tolerated prob. damaging
pos. new donor site
BS1 LB
PM2 was applied when a variant was absent or extremely rare (<0.004%) in large population cohorts, as proposed by ClinGen’s Inherited Cardiomyopathy Expert Panel (Kelly et al., 2018: PMID 29300372). Variants in autosomal recessive genes were classified as likely benign as they were reported >5 times in homozygous state in gnomAD. ACMG=American College of Medical Genetics,
CADD=Combined Annotation Dependent Depletion v1.4, gnomAD=Genome Aggregation Database v2.1.1, HGVS=Human Genome Variation Society, PolyPhen-2=Polymorphism Phenotyping v2, prob.=probably, pos.=possible, SIFT=Sorting tolerant from intolerant, NA=not available/unknown, LB=likely benign, VUS=variant of uncertain significance, LP=likely pathogenic.
